Transfection of highly metastatic BL6 melanoma cells with H-2Kb gene reversed their metastatic ability. Loss of metastatic properties by H- 2Kb+ cells seems to be related to involvement of immunological and some nonimmunological mechanisms that might be associated with H-2Kb gene- induced alterations in melanoma cell properties. Indeed, we found that expression of H-2Kb gene in BL6 melanoma cells altered various cell characteristics such as cell surface carbohydrates with appearance of alpha-galactosyl epitopes reacting with GS1B4 lectin, reduction in sialylation and unmasking SBA lectin binding cell membrane carbohydrates, loss of melanoma associated antigen (MAA) and B-tropic ecotropic retrovirus specific for melanomas of C57BL/6 origin. These nonimmunological effects were induced by class I H-2Kb or H-2Kd, but not H-2Db, H-2Dd or H-2Ld or class II H-2IAk genes. Lack of alpha-galactosyl epitopes in BL6 melanoma is a result of down-regulation of the alpha 1,3 galactosyltransferase (alpha 1,3GT) gene that was up-regulated in melanoma cells expressing the endogenous or transfected H-2Kb gene. The main objective of our proposal is to identify the H-2Kb gene-induced phenotypic changes responsible for reversal of the metastatic ability of murine melanoma cells. These studies are based on the following working hypothesis: Alterations in cell membrane glycosylation induced by H-2Kb gene are responsible for reduction of metastatic properties of BL6 melanoma cells. H-2Kb gene expression activates alpha 1,3GT gene and leads to production of alpha 1,3GT that compete with alpha 2,3 sialyltransferase (alpha 2,3ST) or alpha 2,6 sialyltransferase (alpha 2,6ST) for capping of the N-acetyllactosamine carbohydrate chain in the Golgi apparatus. As a result, cell membranes of H-2Kb+ BL6 cells become galactosylated and less sialylated, reducing their ability to survive in circulation and develop metastases. To test this hypothesis the following studies are proposed: 1) To test whether the expression of alpha 1,3GT gene and alpha-galactosyl epitopes is associated with concomitant reduction in cell membrane sialylation and inversely correlates with the metastatic ability of various melanomas and lymphomas. 2) To transfect alpha 1,3GT cDNA into alpha 1,3GT negative BL6, JB/MS or JB/RH melanoma cells and to study its effect on expression of alpha-galactosyl epitopes and cell membrane sialylation, activity of alpha 2,3ST and alpha 2,6ST and their metastatic ability. To identify the major glycoprotein and glycolipids expressing alpha-galactosyl epitopes on BL6 melanoma cells transfected with alpha 1,3GT gene. In parallel, to transfect BL6 melanoma cells with alpha 1,2 fucosyltransferase cDNA, in order to compare its ability to reduce cell membrane sialylation and affect metastatic ability. 3) To analyze the mechanisms responsible for alteration of the metastatic potential of these tumor cells by testing their ability to survive in the circulation, their adhesive and invasive properties. This study is a first attempt to obtain direct evidence on the role of cell surface carbohydrates in metastasis formation, using transfection of the gene coding cell surface carbohydrates.
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